Spinach leaf chloroplasts isolated in isotonic media (330 millimolar sorbitol, -1.0 megapascals osmotic potential) had optimum rates of photosynthesis when assayed at -1.0 megapascals. When chloroplasts were isolated in hypertonic media (720 millimolar sorbitol, -2.0 megapascals osmotic potential) the optimum osmotic potential for photosynthesis was shifted to -1.8 megapascals and the chloroplasts had higher rates of CO(2)-dependent O(2) evolution than chloroplasts isolated in 330 millimolar sorbitol when both were assayed at high solute concentrations.Transfer of chloroplasts isolated in 330 millimolar sorbitol to 720 millimolar sorbitol resulted in decreased chloroplast volume but this shrinkage was only transient and the chloroplasts subsequently swelled so that within 2 to 3 minutes at 20 degrees C the chloroplast volume had returned to near the original value. Thus, actual steady state chloroplast volume was not decreased in hypertonic media. In isotonic media, there was a slow but significant uptake of sorbitol by chloroplasts (10 to 20 micromoles per milligram chlorophyll per hour at 20 degrees C). Transfer of chloroplasts from 330 millimolar sorbitol to 720 millimolar sorbitol resulted in rapid uptake of sorbitol (up to 280 micromoles per milligram chlorophyll per hour at 20 degrees C) and after 5 minutes the concentration of sorbitol inside the chloroplasts exceeded 500 millimolar. This uptake of sorbitol resulted in a significant underestimation of chloroplast volume unless [(14)C]sorbitol was added just prior to centrifuging the chloroplasts through silicone oil. Sudden exposure to osmotic stress apparently induced a transient change in the permeability of the chloroplast envelope since addition of [(14)C]sorbitol 3 minutes after transfer to hypertonic media (when chloroplast volume had returned to normal) did not result in rapid uptake of labeled sorbitol.It is concluded that chloroplasts can osmotically adjust in vitro by uptake of solutes which do not normally penetrate the chloroplast envelope, resulting in a restoration of normal chloroplast volume and partially preventing the inhibition of photosynthesis by high solute concentrations. The results indicate the importance of matching the osmotic potential of isolation media to that of the tissue, particularly in studies of stress physiology.